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A new closed-type visual nucleic acid detection method coupled with nucleic acid amplification reaction, nucleic acid invasion reaction and nanoparticle color reaction

A technology of nucleic acid invasion reaction and nucleic acid amplification reaction, which is applied in the field of molecular biology, can solve the problems of cross-contamination of amplification products, low sensitivity, and low discrimination, and achieve low-cost, high-sensitivity, and high-specificity effects

Active Publication Date: 2017-05-17
NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, detection technology based on nucleic acid has been widely used in many aspects such as clinical diagnosis, environmental monitoring, and prevention and control of infectious diseases, such as polymerase chain reaction technology (Polymerase Chain Reaction, PCR) technology, which can realize template Exponential amplification, magnification of millions of times, but this type of technology is usually limited by specificity, so that it cannot be satisfied with the detection of some samples that distinguish single-base target nucleic acids. For example, when detecting gene mutation samples, it was developed by PCR. The real-time PCR (Real Time PCR, RT-PCR) technology not only has limited specificity, low discrimination, requires special equipment, but also has high detection cost.
The nucleic acid invasion reaction based on signal amplification relies on a signal amplification reaction of a special endonuclease. This reaction not only has good specificity, but also has the advantages of constant temperature and low reaction cost. At present, there is a nucleic acid invasion reaction based on Technology coupled with bio-nano-gold technology for molecular detection (see the patent "cascade invasion signal amplification reaction combined with nano-gold-oligonucleotide probe visual nucleic acid detection method", patent application number "201110161736.3"), but due to the The technical sensitivity is low, and only 6×10 template content can be detected 4 Samples with more than one copy / μL still cannot meet the requirements of most clinical samples for direct detection
Therefore, in the detection process of the actual sample, it is necessary to pre-amplify the template to be tested, take out the amplified product after opening the tube, and then perform the nucleic acid invasion reaction and the bionano gold color reaction; but take out the amplification product after opening the tube. The product is very easy to cause cross-contamination of the amplification product, resulting in false positive results

Method used

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  • A new closed-type visual nucleic acid detection method coupled with nucleic acid amplification reaction, nucleic acid invasion reaction and nanoparticle color reaction
  • A new closed-type visual nucleic acid detection method coupled with nucleic acid amplification reaction, nucleic acid invasion reaction and nanoparticle color reaction
  • A new closed-type visual nucleic acid detection method coupled with nucleic acid amplification reaction, nucleic acid invasion reaction and nanoparticle color reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 The single-tube closed-tube visual nucleic acid detection method using nucleic acid amplification reaction combined with cascade nucleic acid invasion reaction and nano-gold color detection to detect ultraviolet quantitative genomic DNA.

[0063] The whole genome DNA is used as a template for detection after gradient dilution to investigate the sensitivity of the method of the present invention.

[0064] Reaction conditions:

[0065] The reaction system in this step is composed of 10mM Tris-HCl (pH7.5), 0.05% Tween-20, 0.05% NonidetP40, and 4mM MgCl 2 , 30mM NaCl, 0.5μM upstream primer (sequence: 5'-CCG GGA GTT GGG CGA GTA C-3', Tm value 70.8℃, SEQ ID NO.1), 0.5μM downstream primer (sequence: 5'- GCC CCC AGC AGG TCC C-3', Tm value is 71.8℃, SEQ ID NO.2), 0.2μM upstream probe (sequence is: 5'-CTC TGC CAC CAG CTC CCA-3', Tm value is 68.5℃ , SEQ ID NO.3), 0.2μM downstream probe (the wild-type probe sequence is: 5'-CGC GCC GAGGCG AAC TTG GGC GAG-C 3 -3’, Tm value is 62....

Embodiment 2

[0067] Example 2 A single-tube closed-tube visual nucleic acid detection method using nucleic acid amplification reaction combined with cascade nucleic acid invasion reaction and nanoprobe color detection is used to detect SNP site typing.

[0068] The detection of SNP site typing using the present invention requires the corresponding downstream probes to be designed for different types of detection sites, and the detection is carried out in two tubes. When the reaction system contains a template that matches the downstream probe, it hybridizes with the nanoprobe When the reaction system does not contain a template that matches the downstream probe, it will aggregate after hybridization with the nanoprobe, thereby realizing the typing of SNP sites.

[0069] Reaction conditions:

[0070] The reaction system in this step is composed of 10mM Tris-HCl (pH7.5), 0.05% Tween-20, 0.05% NonidetP40, and 4mM MgCl 2 , 30mM NaCl, 0.5μM upstream primer (sequence: 5'-CCG GGA GTT GGG CGA GTA C-3', T...

Embodiment 3

[0072] Example 3 The single-tube closed tube of nucleic acid amplification reaction combined with cascade nucleic acid invasion reaction and nano-gold color detection visualizes the effect of different storage time on nano-gold precipitation after nucleic acid detection.

[0073] The whole genome DNA is used as a template for detection, and after the detection reaction is completed, it is placed at room temperature for different times to investigate the precipitation of the nano-gold aggregation reaction in the method of the present invention.

[0074] Reaction conditions:

[0075] The reaction system in this step is composed of 10mM Tris-HCl (pH7.5), 0.05% Tween-20, 0.05% NonidetP40, and 4mM MgCl 2 , 30mM NaCl, 0.5μM upstream primer (sequence: 5'-CCG GGA GTT GGG CGA GTA C-3', Tm value 70.8℃, SEQ ID NO.1), 0.5μM downstream primer (sequence: 5'- GCC CCC AGC AGG TCC C-3', Tm value is 71.8℃, SEQ ID NO.2), 0.2μM upstream probe (sequence is: 5'-CTC TGC CAC CAG CTC CCA-3', Tm value is 68.5...

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Abstract

The invention discloses a novel closed type nucleic acid visual detecting method for coupling a nucleic acid amplification reaction, a nucleic acid intrusive reaction and a nano-particle chromogenic reaction. The detection method comprises the following steps: 1) target nucleic acid amplification and signal conversion phase: further amplifying the target nucleotide sequence, and converting the detection of the target nucleic acid to the detection of a hairpin probe fragment; 2) nanoprobe hybridization phase: after the amplification reaction, under the corresponding conditions, generating a gathering or dispersing phenomenon by the special nanoprobe, and utilizing the phenomenon to realize distinguishing detection to the target nucleic acid. With adoption of the detection method disclosed by the invention, the nucleic acid target sequence can be detected; furthermore, the nucleic acid intrusive reaction is high in specificity, and the nucleic acid sequence with single different basic groups near the intrusive loci can be distinguished and detected, so that the method not only can be used for detecting a template, but also can be used for single basic group distinguished SNP genotyping and gene mutation.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and relates to a new closed visual nucleic acid detection method that couples nucleic acid amplification reaction, nucleic acid invasion reaction and nanoparticle color reaction. Background technique [0002] At present, nucleic acid-based detection technology has been widely used in clinical diagnosis, environmental monitoring, and the prevention and control of infectious diseases, such as polymerase chain reaction (Polymerase Chain Reaction, PCR) technology, which can realize template detection. Exponential amplification, magnification by a million times, but this type of technology is usually not satisfied with the detection of some samples that distinguish single-base target nucleic acids due to specific limitations. For example, when detecting samples with genetic mutations, PCR is developed Real-time PCR (RT-PCR) technology is not only limited in specificity, low in discrimination, requires spec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 周国华王建平邹秉杰
Owner NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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